Animal cell culture is a basic technique in the fields of biology and medicine. The production of living cells in vitro, in the laboratory, permits numerous applications that would be difficult or impossible in vivo, in the living animal. The culture of animal cells requires a defined medium containing specific quantities of certain chemicals, and in addition for most cells, up to 15% of an undefined nutrient medium usually fetal bovine serum (FBS). Serum from newborn calves and other mammals is also used, but FBS is preferred because of its high level of growth factors and low cross-reactivity with other animal cells. FBS or other mammalian products are also used to coat the surface of culture-ware to promote cell attachment.
The production of FBS in this country is an estimated 700,000 liters annually worth $300 to $400 million. The industry obtains fetal calves for bleeding from slaughter houses, or in some cases, rears herds of cattle for this purpose. These herds are held in as isolated a situation as possible in order to prevent disease. Whole blood is obtained aseptically (by syringe) from an animal, centrifuged to separate cells from serum, and the serum filtered to 0.22 microns to remove most bacteria. Often, serum is heated to 56.degree. C. to inactivate the complement system, a group of immune proteins.
Contamination of cell cultures because of infectious organisms in serum can be a serious problem. Bacteria, fungi, viruses and mycoplasma have been isolated from bovine serum. In the period from 1960 1980, mycoplasma from bovine serum was the second major group of contaminants found in cell culture (Barile, 1977). Now, FBS is usually screened for mycoplasma and most viruses. However, a more serious cause for concern is an all-protein infectious agent called a prion for which no test is available (Kingman, 1993). This organism causes a fatal brain disease in mammals called Bovine Spongiform Encephalopathy (BSE), or "mad cow disease". BSE occurs in sheep, cows, and other mammals, and is most likely the cause of similar neuro-degenerative diseases in humans. In Britain in 1986, BSE resulted in the destruction of cattle and fears for the safety of the meat supply and other bovine products. Since then, the disease has turned up in cattle in many other countries. Consequently, serum from these countries cannot be imported for use in the U.S.
Basic texts in cell culture teach a like-for-like approach, or mammalian sera, especially FBS, for culture of mammalian cells (Freshney, 1987). So firmly established is this dogma of FBS, that some cell culture publications refer only to "serum" when FBS is intended (Pollard and Walker, 1984). Likewise, major serum suppliers such as Sigma Chemical Co. and Hy-Clone Laboratories include only bovine and some equine sera in their cell culture catalogs. In practice during the past thirty years, only mammalian sera have been used for the culture of mammalian cells.
U.S. Pat. No. 4,449,480 to Isom et al. discloses freshwater mussels in an artificial habitat utilizing growth media. However, the Isom et al. invention is directed to larviculture, that is, the provision of food and habitat for the larval stage of young animals such as aquatic invertebrates. Isom et al. are not concerned with cell culture. Larviculture is an interim technique, having the goal of keeping the larval animal alive until it can progress to the next stage, or in this particular case, until it can transform from a parasitic stage and feed independently on its own (Pennack 1953). Thus, the Isom et al. invention is not applicable to the instant problem, as cell lines do not grow and transform to become independent of their culture medium and learn to feed on their own.
Further, the method taught by Isom et al. can only operate on the parasitic stage of whole, multicellular animals (Barnes, 1963), and is not applicable to cell lines derived from the organs and tissues of a whole animal. A fundamental purpose of cell culture is to permit processes and experimentation in cells that would not be possible to perform on a whole animal. Isom et al. borrow certain techniques from cell culture (for example, a modified solution or medium of salts and proteins) and from bacteriology (for example, antibiotics). The use of these tools, however, does not render the Isom et al. method a cell culture or bacteriology process, and the disclosed method cannot function as such. Thus, the Isom et al. method cannot provide a solution to the instant problem.